Peptide fructose and protein conjugate with the same

ABSTRACT

It is intended to provide an antibody specific to HbA1c, antibody-producing cells capable of supplying the antibody in a stable state in the future, and a method of constructing the antibody-producing cells without any probability factors, and a method which comprises fusing mouse spleen cells, which have been sensitized with an immunogen composed of a compound containing the following structural formula (I) and a binding protein, with a myeloma-origin cell line, obtaining monoclonal antibody-producing cells by cloning, and then purifying and acquiring the monoclonal antibody produced by these cells into the culture supernatant

RELATED APPLICATIONS

This is a US national phase filing under 35 U.S.C. § 371 ofPCT/JP1/06110 filed Jul. 13, 2001 and claims priority from JP2000-214053 filed Jul. 14, 2000 and JP 2000-350979 filed Nov. 17, 2000.

TECHNICAL FIELD

The present invention relates to a peptide fructose compound, which canbe used as an immunogen for production of antibodies for hemoglobin A1c(hereinafter abbreviated as HbA1c). The present invention also relatesto a protein conjugate of the compound and a protein, and an antiserumand an antibody obtained using the peptide fructose-protein conjugate.

BACKGROUND ART

Conventionally, as an immunogen required for production of anti-HbA1cantibodies, HbA1c itself has been generally used. HbA1c has a structuresimilar to that of hemoglobin A0 (hereinafter abbreviated as HbA0) whichconstitutes 90% of the total amount of hemoglobin. Whereas HbA0 has nosugar chain at the N-terminus of its β chain, HbA1c has a fructosebinding to the N-terminus of its β chain. This is the only differencebetween HbA0 and HbA1c. Therefore, most of the antibodies which areproduced using HbA1c itself as an immunogen can also recognize HbA0.Among the antibodies for hemoglobin, there are only a small number ofantibodies capable of recognizing only HbA1c. Conventionally, such agroup of antibodies have been screened for those that bind to HbA1c.

Such screening work is labor-intensive and costly. Therefore, it may beconceived to use an epitope capable of recognizing only HbA1c. However,in most cases, an epitope alone does not have sufficient antigenicity orimmunogenicity in production of an antiserum or antibodies. Therefore,an animal is immunized with an epitope in conjunction with an adjuvantor a carrier. However, immunization of an animal with a carrier and anepitope is conventionally labor-intensive and is not necessarilyefficient to obtain an intended antiserum or antibodies, and is alsocostly. Moreover, acquisition of an intended antibody depends onprobabilistic factors, and the above-described method cannot be said tobe reliable. No monoclonal antibody without cross reactivity to HbA0 hasbeen obtained, and there has been conventionally no established methodwith which such a monoclonal antibody can be reliably and simplyproduced.

In order to solve the above-described problems, an object of the presentinvention is to provide a peptide fructose compound and a proteinconjugate which are immunogens capable of producing anti-HbA1cantibodies, preferably only anti-HbA1c antibodies, where theprobabilistic factors are removed and the antibodies have no crossreactivity to HbA0. Another object of the present invention is toprovide an antiserum or antibodies produced using such a peptidefructose compound or a protein conjugate. Still another object of thepresent invention is to provide a method of producing anantibody-producing cell capable of supplying an antibody specific toHbA1c and without cross reactivity to HbA0 and capable of stablysupplying such an antibody for future use, where the probabilisticfactors are removed.

DISCLOSURE OF THE INVENTION

In order to achieve the above-described objects, the present inventionprovides the following.

In one aspect, the present invention provides a peptide fructosecompound represented by formula (I) below:

wherein R1 represents any molecule having an —SH group, the R1 is linkedto the carboxy-terminus of R2 at (b) with a covalent bond, the R2contains one or more amino acids derived from the amino acid sequence ofHbA1 or an amino acid analog functionally equivalent to the amino acids,and the R2 is linked to the fructose at the amino-terminus thereof.

Here, the molecule having an —SH may be any molecule known in the art.Examples of such a molecule include, but are not limited to, cysteineand homocysteine. The linkage (b) is usually made by a covalent bond,and may be made by any type of bond (e.g., a hydrogen bond) other than acovalent bond as long as the bond has an ability to induce an antibodyor an antiserum. The covalent bond may be any type of covalent bond, andpreferably a peptide bond (amide bond). Moreover, instead of theabove-described fructose, any molecule (e.g., other sugars) functionallyequivalent thereto may be used.

In another embodiment, the R2 may contain at least a peptide representedby formula (II) below:

In one embodiment, the R2 may contain at least a peptide represented byformula (III) below:

In another embodiment, the R1 may contain at least one cysteine residue.In another embodiment, the R1 may comprise a peptide or a peptideanalog. In another embodiment, the R1 may comprise a peptide. In anotherembodiment, the R1 may be a cysteine residue.

In another embodiment, the covalent bond at (b) is a peptide bond.

In another aspect, the present invention provides a protein conjugate,wherein a peptide fructose compound according to any one of claims 1 to8 is linked with a protein. Preferably, this protein may not behemoglobin, or may be hemoglobin which has no cross reactivity tohemoglobin A1c.

In one embodiment, the protein is selected from the group consisting ofbovine serum albumin (BSA), chicken-γ globulin (CGG), and Keyhole LimpetHemocyanin (KLH). Preferably, the protein is CGG.

In another aspect, the present invention provides an antiserum, producedin the blood of an animal by injecting a peptide fructose compound ofthe present invention or a protein conjugate of the present invention.

In another aspect, the present invention provides an antibody, isolatedfrom an antiserum of the present invention.

In another aspect, the present invention provides a monoclonalantibody-producing cell, wherein the monoclonal antibody-producing cellis obtained by fusing a spleen cell of a mouse sensitized with a peptidefructose compound of the present invention or a protein conjugate of thepresent invention, with a myeloma-derived cell, and cloning a fusedcell, and the monoclonal antibody-producing cell produces a monoclonalantibody capable of specifically binding to human hemoglobin A1c. In oneembodiment, the present invention provides a monoclonalantibody-producing cell designated as deposit number FERM BP-7637 orFERM BP-7636.

In another aspect, the present invention provides a method of producinga monoclonal antibody-producing cell capable of producing a monoclonalantibody capable of specifically binding to human hemoglobin A1c. Themethod comprises the steps of:

a) sensitizing a mouse with a peptide fructose of the present inventionor a protein conjugate of the present invention; and

b) isolating a spleen cell from the sensitized mouse, and fusing thespleen cell with a myeloma-derived cell.

In another aspect, the present invention provides a monoclonal antibody,produced by a monoclonal antibody-producing cell of the presentinvention, which specifically binds to human hemoglobin A1c.

In one embodiment, the binding constant to human hemoglobin A1c thereofmay be 10⁴ or more. Preferably, the binding constant may be 10⁵ or more,10⁶ or more, 10⁷ or more, 10⁸ or more, 10⁹ or more, or 10¹⁰ or more.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an immunizing schedule used in one embodiment of thepresent invention.

FIG. 2 is a diagram showing the performance assessment of an antiserumfor one embodiment (F-CGG) of the present invention.

FIG. 3 is a diagram showing the performance assessment of an antiserumfor one embodiment (F-KLH) of the present invention.

FIG. 4 is a diagram showing the antibody titer of an antiserum 77 daysafter immunization according to one embodiment of the present invention.

FIG. 5 is a diagram showing the antibody titer of a monoclonal antibody(4F) according to one embodiment of the present invention.

FIG. 6 is a diagram showing the antibody titer of a monoclonal antibody(8E) according to one embodiment of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, some terms used herein will be described. It should benoted that unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

The term “peptide fructose compound” refers to a compound comprising apeptide and fructose. A typically peptide fructose compound has thefollowing structure:

where R1 represents any molecule having an —SH group; the R1 is linkedto the carboxy terminus of R2 by covalent bond at (b); and R2 includesone or more amino acids derived from HbA1 or amino acid analogsfunctionally equivalent to the amino acids, and the R2 is linked tofructose at the amino-terminus thereof.

The term “peptide”, “oligopeptide”, “polypeptide” and “protein” are usedinterchangeably to refer to a polymer of two or more amino acids(naturally occurring or non-naturally occurring) with peptide bonds.

The term “amino acid” refers to an organic compound having an aminogroup (—NH₂) and a carboxy group (—COOH) in the same molecule, and asused in the art, also includes imino acids having an imino group, suchas proline and hydroxyproline. Amino acids used herein may benaturally-occurring amino acids (asparagine (hereinafter abbreviated asAsn), aspartic acid (hereinafter abbreviated as Asp), alanine(hereinafter abbreviated as Ala), arginine (hereinafter abbreviated asArg), isoleucine (hereinafter abbreviated as Ile), glycine (hereinafterabbreviated as Gly), glutamine (hereinafter abbreviated as Gln),glutamic acid (hereinafter abbreviated as Glu), cysteine (hereinafterabbreviated as Cys), serine (hereinafter abbreviated as Ser), tyrosine(hereinafter abbreviated as Tyr), tryptophan (hereinafter abbreviated asTrp), threonine (hereinafter abbreviated as Thr), valine (hereinafterabbreviated as Val), histidine (hereinafter abbreviated as His),phenylalanine (hereinafter abbreviated as Phe), proline (hereinafterabbreviated as Pro), methionine (hereinafter abbreviated as Met), lysine(hereinafter abbreviated as Lys), and leucine (hereinafter abbreviatedas Leu)), or non-naturally occurring amino acids. The amino acid may bean α-amino acid, β-amino acid, γ-amino acid, δ-amino acid, ω-amino acid,or the like. The amino acid may be of the L type or D type, and ispreferably of the L type.

The term “non-naturally occurring amino acid” refers to an amino acidwhich is not found in a naturally-occurring protein. Examples of thenon-naturally occurring amino acid include norleucine,para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine,3-amino-2-benzylpropionic acid, D- or L-homoarginine, andD-phenylalanine. The non-naturally occurring amino acid also includesones with an —SH group, such as homocysteine.

The term “amino acid analog” refers to a molecule having a physicalproperty or a function similar to that of an amino acid, but is not anamino acid itself. Examples of amino acid analogs include ethionine,canavanine, and 2-methylglutamine. The similarity of such a physicalproperty or function of an amino acid analog may be determined based onwhether or not the linkage of the analog to other compounds issubstantially the same as that of the amino acid, as described herein.

For a peptide fructose compound of the present invention, amino acidsubstitution and the like, with substantially no change infunctionality, is performed by chemical synthesis or by changing a codoncoding an amino acid in a DNA sequence using genetic engineeringtechniques. The present invention is not so limited.

A certain amino acid may be substituted with another amino acid in aprotein structure, for example, a cationic region or a binding site fora substrate molecule, without significant reduction or loss ofinteractive binding capability. The biological function of a certainprotein is determined by the interaction capability and properties ofthe protein. Therefore, even if substitution of a particular amino acidis performed in an amino acid sequence (or at the DNA code sequencelevel), a protein may maintain its original properties after thesubstitution. Therefore, peptides disclosed herein or DNA encoding thepeptides may be modified in various manners without clearly impairingtheir biological utility.

When the above-described modifications are designed, the hydrophobicityindex of amino acids may be taken into consideration. The hydrophobicamino acid index plays an important role in providing a protein with aninteractive biological function, which is generally recognized in theart (Kyte. J and Doolittle, R. F., J. Mol. Biol. 157(1):105–132, 1982).The hydrophobic property of an amino acid contributes to the secondarystructure of a generated protein and then regulates interactions betweenthe protein and other molecules (e.g., enzymes, substrates, receptors,DNA, antibodies, antigens, etc.). Each amino acid is given ahydrophobicity index based on the hydrophobicity and charge propertiesthereof as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamic acid (−3.5); glutamine (−3.5); aspartic acid (−3.5); asparagine(−3.5); lysine (−3.9); and arginine (−4.5)).

It is well known that if a certain amino acid is substituted withanother amino acid having a similar hydrophobicity index, a resultantprotein may still have a biological function similar to that of theoriginal protein (e.g., a protein having an equivalent enzymaticactivity). For such an amino acid substitution, the hydrophobicity indexis preferably within ±2, more preferably within ±1, and even morepreferably within ±0.5. It is understood in the art that such an aminoacid substitution based on hydrophobicity is efficient. As described inU.S. Pat. No. 4,554,101, amino acid residues are given the followinghydrophilicity indexes: arginine (+3.0); lysine (+3.0); aspartic acid(+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2);glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1);alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3);valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5); and tryptophan (−3.4). It is understood that anamino acid may be substituted with another amino acid which has asimilar hydrophilicity index and can still provide a biologicalequivalent. For such an amino acid substitution, the hydrophilicityindex is preferably within ±2, more preferably within ±1, and even morepreferably within ±0.5.

The term “conservative substitution” as used herein refers to amino acidsubstitution in which a substituted amino acid and a substituting aminoacid have similar hydrophilicity indexes or/and hydrophobicity indexes.Examples of conservative substitutions, which are well known to thoseskilled in the art, include, but are not limited to, substitutionswithin each of the following groups: arginine and lysine; glutamic acidand aspartic acid; serine and threonine; glutamine and asparagine; andvaline, leucine, and isoleucine.

Herein, in order to produce functionally equivalent peptide fructosecompounds, amino acid addition, deletion, substitution, or modificationcan be performed. Amino acid substitution refers to substitution of oneor more amino acids in an original peptide, for example, 1 to 10 aminoacids, preferably 1 to 5, and more preferably 1 to 3. Amino acidaddition refers to addition of one or more amino acids to an originalpeptide chain, for example, 1 to 10 amino acids, preferably 1 to 5, andmore preferably 1 to 3. Amino acid deletion refers to deletion of one ormore amino acids from an original peptide, for example, 1 to 10 aminoacids, preferably 1 to 5, and more preferably 1 to 3. Amino acidmodification includes, but is not limited to, amidation, carboxylation,sulfation, halogenation, alkylation, glycosylation, phosphorylation,hydration, acylation (e.g., acetylation), and the like. An amino acidfor substitution or addition may be a naturally-occurring amino acid oralternatively a non-naturally-occurring amino acid. Anaturally-occurring amino acid is preferable.

The term “peptide analog” refers to a compound equivalent to a peptidewith respect to at least one chemical or biological function, but not apeptide itself. Therefore, a peptide analog includes a peptide in whichone or more amino acid analogs have been added to the original peptideor one or more amino acids have been substituted with an amino acidanalog. A peptide analog has such additions or substitutions in which afunction of the peptide is substantially the same as that of theoriginal peptide (e.g., a similarity of pKa values, a similarity offunctional groups, a similarity of linkage with other molecules, asimilarity of water solubility, and the like). Such a peptide analog canbe produced using techniques well known in the art.

In one aspect, a peptide fructose of the present invention is a peptidefructose compound represented by the following formula (I).

A peptide fructose compound typically comprises an introduced moietyhaving an —SH group (e.g., a cysteine residue) in addition tofructose-valine-histidine which is a characteristic structure of HbA1c,which makes it easy to bind to a protein. Therefore, a protein conjugatecan be produced by binding a peptide fructose compound to a protein.

A protein conjugate of the present invention is characterized in that apeptide fructose compound represented by formula (I) is linked with aprotein excluding hemoglobin. The protein conjugate typically hasfructose-valine-histidine, which is a characteristic structure of HbA1c,and a protein excluding hemoglobin, and thus, does not have a structurecommon to hemoglobin HbA0. Therefore, the protein conjugate can be usedas an immunogen to reliably produce an anti-HbA1c antibody without crossreactivity to hemoglobin HbA0, and preferably without cross reactivityto hemoglobins other than HbA1c.

In a peptide fructose-protein conjugate, a peptide fructose compoundrepresented by formula (I) is linked to a protein, excluding hemoglobin,via an —SH group, preferably via a disulfide bond. The peptide fructosecompound can be easily linked to a protein via a thiol group (functionalgroup) such as cysteine to easily produce a protein conjugate. Moreover,the amount of the peptide fructose compound reacting with a protein canbe controlled and the amount of the peptide fructose compound introducedinto a protein can be measured. Another advantage of the introduction ofan —SH group is formation of a relatively stable disulfide (S—S) bond,for example.

As the protein, any protein other than hemoglobin can be used withoutparticular limitation. Examples of the protein include bovine serumalbumin (hereinafter abbreviated as BSA), chicken-γ globulin(hereinafter abbreviated as CGG), and Keyhole Limpet hemocyanin(hereinafter abbreviated as KLH).

In formula (I), R2 preferably includes a residue represented by thefollowing formulas (II) or (III).

The peptide fructose compound has an introduced structure,leucine-threonine, which is a characteristic structure of HbA1c, inaddition to fructose-valine-histidine. This peptide fructose compoundcan be used to produce an anti-HbA1c antibody which has a higher levelof ability to bind to HbA1c and/or a high level of selectivity to HbA1c.

In formula (I), when R2 contains a number of amino acids, whichconstitute a characteristic structure of HbA1c, or multimers of theamino acids, an anti-HbA1c antibody having a greater binding strength toHbA1c can be preferably produced. Note that when the number of the aminoacid multimers which are the characteristic structure of HbA1c isexcessively large, the cross reactivity of the obtained anti-HbA1cantibody to hemoglobins other than HbA1c is likely to be high.Therefore, the number of amino acid multimers which are thecharacteristic structure of HbA1c in R1 and R2 is adjusted from aviewpoint of the binding strength, and the cross reactivity tohemoglobins other than HbA1c, of the obtained anti-HbA1c antibody.

Here, a protein, such as CGG, is preferably used as a protein forproducing a protein conjugate of the present invention. CGG is a proteinwhich has no similarity to murine protein and a high level of capabilityto induce immunity. CGG also has a high level of solubility, which isanother reason that CGG is appropriate for use in the present invention.A peptide fructose compound of the present invention can be introducedinto such a protein at a rate of at least 10 peptide fructose compoundsper protein, preferably at least 20 peptide fructose compounds perprotein, more preferably at least 25 peptide fructose compounds perprotein, and even more preferably at least 30 per protein. Byintroducing a number of peptide fructose compounds of the presentinvention per protein in this manner, it is possible to produceantiserum or antibodies more efficiently.

As a reagent used for linking a peptide fructose compound with aprotein, any compound having a functional group capable of reacting witha thiol group of a residue, such as cysteine having a thiol group, and afunctional group capable of reacting with an amino group or carboxygroup in the protein, can be used. Examples of such a compound includeN-γ-maleimidebutyryloxy-succinimide ester (hereinafter abbreviated asGMBS), succinimidyl 4-(N-maleimidemethyl)cyclohexane-1-carboxylate(hereinafter abbreviated as SMCC), succinimidyl4-(p-maleimidephenyl)butylate (hereinafter abbreviated as SMPB),4-succinimidyloxycarbonylmethyl-α-(2-pyridyldithio)tolu ene (hereinafterabbreviated as SMPT), andO-succinimidyl-3-(2-pyridyldithio)-1-propionate (hereinafter abbreviatedas SPDP). Among them, one which is easily available and can be used tomeasure the number of peptide fructoses introduced into a protein, ispreferable. Such a reagent is SPDP, for example.

An antiserum of the present invention is characterized in that theantiserum is produced in the blood of an animal into which theabove-described peptide fructose-protein conjugate has beenadministered. The antiserum has less cross reactivity to hemoglobinsother than HbA1c and can recognize the characteristic structure ofHbA1c.

As an animal for producing an antiserum, any conventional animal forimmunization can be used without particular limitation. Example of suchan animal include mice, rats, rabbits, goats, sheep, and horses. Also,by producing hybridomas using spleen cells from a mouse immunized with apeptide fructose-protein conjugate of the present invention, ananti-HbA1c monoclonal antibody can be produced. An antibody of thepresent invention may be humanized. A method of humanizing an antibodyis well known in the art.

In order to achieve the above-described objects, a monoclonal antibodyof the present invention is produced by fusing a spleen cell, which isderived from a mouse sensitized with an immunogen comprising a conjugateof a peptide fructose compound having a structure represented by formula(I) and a protein, with a cell line derived from myeloma, followed bycloning, and allowing a monoclonal antibody-producing cell obtained bycloning to produce the monoclonal antibody in a supernatant of culturemedium.

A monoclonal antibody-producing cell of the present invention may beobtained by fusing a spleen cell, which is derived from a mousesensitized with an immunogen comprising a peptide fructose and aprotein, with a myeloma-derived cell line, followed by cloning.

A monoclonal antibody of the present invention is produced by a fusioncell (hybridoma) of a spleen cell, which is derived from a mouseimmunized with a peptide fructose compound of the present invention or aprotein conjugate therewith as an immunogen, with a myeloma-derivedcell. However, a supernatant from a culture in which fusion cells(hybridomas) have been grown can be purified using an antibodypurification column filled with protein A Sepharose gel or the like toobtain an antibody of interest.

A peptide fructose compound of the present invention hasfructose-valine-histidine which is a characteristic structure of HbA1c.The peptide fructose compound linked with a protein is an effectiveimmunogen for obtaining an anti-HbA1c antibody.

A monoclonal antibody-producing cell of the present invention can beobtained as follows: a spleen cell, which is derived from a mouseimmunized as described above, is fused with, for example, amyeloma-derived cell, such as p3×63·Ag8·653 using a fusion acceleratingreagent, such as polyethylene glycol, and a number of the resultantfusion cells are screened for one which produces only an antibody whichreacts with HbA1c but not HbA0 , using an assessment method, such as anenzyme-linked immunosolvent assay (hereinafter abbreviated as ELISA).

EXAMPLES

Hereinafter, the present invention will be described in more detail byway of examples. It should be understood that examples provided belowindicate illustrative embodiments of the present invention, and are notintended to limit the present invention. Note that in the examples, CGGwas used as an exemplary protein.

Example 1 Preparation of Peptide Fructose

In Example 1, a peptide fructose represented by formula (V) below wasprepared by the following procedure.

1. Introduction of Cys Group Into Wang Resin (Solid Phase)

12.95 g (30 mmol) of N-fluorenylmethoxycarbonyl-S-t-butylthiocysteine(hereinafter abbreviated as Fmoc-Cys(StBu)) was dissolved in 50 ml ofchloroform, followed by addition of 3.095 g (15 mmol) ofdicyclohexylcarbodiimide (hereinafter abbreviated as DCC) and stirringat room temperature for 10 minutes. The resultant precipitate(dicyclohexyl urea) was filtered out and a filtrate was dried underreduced pressure. The dried filtrate was dissolved in 50 ml ofdimethylformamide (hereinafter abbreviated as DMF), followed by additionof 10 g of Wang resin (binding OH group: 1 mmol/g) to obtain asuspension. 0.733 g (6 mmol) of dimethylaminopyridine (hereinafterabbreviated as DMAP) was added to the suspension, and stirred at roomtemperature for 60 minutes. The reaction solution was subjected tofiltration and the resin was washed with DMF twice and then chloroformtwice, followed by drying under vacuum overnight to obtain 11.8 g ofdried resin (hereinafter abbreviated Fmoc-Cys(StBu)-Resin).

Thereafter, the degree of introduction of a Cys group into the resin wasmeasured by the following manner. 5 ml of 50% piperidine/DMF mixedsolution was added to 100 mg of the dried resin, and stirred at roomtemperature for 5 minutes. The resin was filtered out and the absorbanceresultant filtrate was measured at 301 nm. The absorbance was 66.4. Theabsorption at 301 nm was derived from fluorenylmethylpiperidine(hereinafter abbreviated as Fmp) generated by piperidine deprotectingthe Fmoc-Cys group introduced into the resin. Therefore, the amount ofreleased Fmp indicates the amount of a Cys group. The Fmp concentration[Fmp] can be calculated by formula (1), where the molar absorbancecoefficient of Fmp at 301 nm is 7800.

$\begin{matrix}\begin{matrix}{\lbrack{Fmp}\rbrack = {66.4/7800}} \\{= {8.513 \times 10^{- 3}\mspace{11mu}(M)}}\end{matrix} & \left( {{Formula}\mspace{20mu} 1} \right)\end{matrix}$

Since the reactive OH group of the resin is 1 mmol/g, the concentrationof the reactive OH group is roughly calculated to be 0.02 M. Therefore,the rate of introduction of the Cys group into the resin can becalculated by formula (2).

$\begin{matrix}\begin{matrix}{\left( {{Ratio}\mspace{14mu}{of}\mspace{14mu}{introduction}\mspace{14mu}{of}\mspace{14mu}{Cys}\mspace{20mu}{groups}} \right) = {8.513 \times {10^{- 3}/0.02`}}} \\{= {0.43\mspace{11mu}\left( {43\;\%} \right)}}\end{matrix} & \left( {{Formula}\mspace{20mu} 2} \right)\end{matrix}$

2. Introduction of His Group Into Fmoc-Cys(StBu)-Resin

10 g (4.3 mmol on a Cys basis) Fmoc-Cys(StBu)-Resin was dispersed in 50ml of 20% piperidine/DMF, and stirred at room temperature for 10minutes. The solvent was removed by filtration and thereafter 20%piperidine/DMF reaction was repeated. The resin was washed with 50 ml ofDMF three times, and a small amount of the resin was subjected to aninhydrin test in a methanol solution. As a result, it was confirmedthat the amino group was in a free state. The resin was dispersed in 20ml of DMF. To this solution, 8.447 g (10.75 mmol) offluorenylmethoxycarbonyltritylhistidine pentafluorophenylester(hereinafter abbreviated as Fmoc-His(Trt)-Opfp) was added, and stirredat room temperature for two hours. The resin was washed with 50 ml ofDMF three times. Thereafter, a small amount of resin was subjected to aninhydrin test in a methanol solution. As a result, it was confirmedthat no free amino group existed. Thereafter, the resin was washed withchloroform three times, followed by drying under vacuum overnight toobtain 11.2 g of the dried resin (hereinafter abbreviated asFmoc-His(Trt)-Cys(StBu)-Resin).

The ratio of introduction of a His group into the resin was measured ina manner as described above. The ratio of introduction of a His groupwas 38%.

3. Introduction of Valine Group (Hereinafter Abbreviated as Val) IntoFmoc-His(Trt)-Cys(StBu)-Resin

11 g (4.2 mmol on a His basis) Fmoc-His(Trt)-Cys(StBu)-Resin wasdispersed in 100 ml of 20% piperidine/DMF, and stirred at roomtemperature for 10 minutes. The solvent was removed by filtration andthereafter 20% piperidine/DMF reaction was repeated. The resin waswashed with 50 ml of DMF three times, and a small amount of the resinwas subjected to a ninhydrin test in a methanol solution. As a result,it was confirmed that the amino group was in a free state. The resin wasdispersed in 20 ml of DMF. To this solution, 5.234 g (10.53 mmol) offluorenylmethoxycarbonyltritylvaline pentafluorophenylester (hereinafterabbreviated as Fmoc-Val-Opfp) was added, and stirred at room temperaturefor two hours. The resin was washed with 50 ml of DMF three times.Thereafter, a small amount of resin was subjected to a ninhydrin test ina methanol solution. As a result, it was confirmed that no free aminogroup existed. Thereafter, the resin was washed with chloroform threetimes, followed by drying under vacuum overnight to obtain 10.0 g of thedried resin (hereinafter abbreviated asFmoc-Val-His(Trt)-Cys(StBu)-Resin).

The ratio of introduction of a Val group into the resin was measured ina manner as described above. The ratio of introduction of a Val groupwas 21%.

4. Removal of Peptide Chain From Resin

9 g of Fmoc-Val-His(Trt)-Cys(StBu)-Resin was dispersed in 100 ml of 20%piperidine/DMF, and stirred at room temperature for 10 minutes. Thesolvent was removed by filtration. Thereafter, the resin was reactedwith 20% piperidine/DMF again. The resin was washed with 100 ml of DMFthree times. A small amount of the resin was subjected to a ninhydrintest in a methanol solution. As a result, it was confirmed that theamino acid group was in a free state. The resin was washed withchloroform three times, and thereafter, was dispersed in 100 ml of 50%chloroform/trifluoroacetic acid (hereinafter abbreviated as TFA) mixedsolvent, and stirred at room temperature for two hours. The resin wasfiltered out, and washed with 100 ml of chloroform/TFA (=4/1) mixedsolvent two times. All of the filtrate and the washing solution werecollected and concentrated under reduced pressure. The residue wasdissolved in a small amount of TFA, and dispersed in about 300 ml ofether, followed by centrifugation. The supernatant was removed and theprecipitate was washed with ether two times, followed by drying undervacuum overnight. As a result, 0.63 g of the crude product (Val-His-Cys)was obtained.

5. Introduction of Fructose Into Peptide Chain Due to AmadoriRearrangement Reaction

500 mg (1.126 mmol) of synthetic peptide (Val-His-Cys) and 270 mg (1.5mmol) of glucose were dissolved in 25 ml of pyridine/acetic acid (=1/1)mixed solvent, and stirred in a dark place at room temperature for 5days. The solvent was removed under vacuum, and thereafter, the residuewas dissolved in 25 ml of water, followed by concentration under vacuum.The concentrate was dissolved in a small amount of water and loaded ontoa Dowex50WX8 column (manufactured by Sigma, diameter 3.5 cm×length 25cm) which was a strongly acidic ion exchange resin. The column waswashed with about 2 L of water to remove the glucose. Thereafter, theproduct was eluted with, 1 N ammonia water, and the eluate waslyophilized. The resultant product was dissolved in 0.1 Mtriethylammonium buffer (pH=8.2), and loaded onto an Affigel 601 column(manufactured by Pharmacia, diameter 1.7 cm×length 20 cm). The columnwas washed with 1 L of 0.1 M triethylammonium buffer (pH=8.2) and then 1L of water. Finally, the product was eluted with 0.1% formic acid, andthe eluate was lyophilized. 50 mg of a crudely purified product ofpeptide fructose (fructose-Val-His-Cys) was obtained. Table 1 showschemical shifts and peak positions of NMR for the product measured indeuterated methanol.

TABLE 1

Peak position Chemical shift Number of Number of (numeral in the (δ,ppm) hydrogens peaks formula above) 1.14 6 4 1 2.20 1 m 2 2.93 2 8 93.22 2 8 5 3.5–3.9 7 m 10–14 3.76 1 2 3 4.60 1 3 8 4.82 1 3 4 7.38 1 1 68.77 1 1 7 Note: m indicates a large number of peaks.

(Preparation of Peptide Fructose-Protein Conjugate)

In this example, CGG was used as a protein.

1. Preparation of pyridyldithiopropionylated CGG (CGG-SPDP))

200 mg (1.33×10⁻³ mmol) of CGG was dissolved in 50 ml of phosphatebuffer saline (hereinafter abbreviated as PBS). 5 ml of SPDP/ethanolsolution (SPDP=40 mg, 0.13 mmol, an equivalent weight of 100) wasdropped into the mixture while stirring. After stirring at roomtemperature for 30 minutes, the resultant precipitate was removed bycentrifugation (10 minutes, 20,000 rpm). The resultant supernatant wassubjected to gel filtration using a Sephadex G25M column (manufacturedby Pharmacia, diameter 2 cm×length 80 cm) to obtain 80 ml ofCGG-SPDP/PBS solution.

The number of SPDPs binding to one CGG molecule was determined asfollows. 1 ml of the resultant CGG-SPDP PBS solution was used and theabsorbance thereof was measured at 280 nm. The absorbance was 3.73.

50 μl of 100 mM dithiothreitol (hereinafter abbreviated as DTT) aqueoussolution was added to the CGG-SPDP/PBS solution, and was allowed tostand for 5 minutes. Thereafter, the absorbance was measured at 343 nm,with a result of 2.80.

Assuming that the molecular absorbance coefficient of pyridine-2-thionereleased by DTT reduction at 343 nm was 8.08×10³, the concentration[pyridine-2-thione] was determined by formula (3).

$\begin{matrix}\begin{matrix}{\left\lbrack {{pyridine}\text{-}2\text{-}{thione}} \right\rbrack = {2.80/\left( {8.08 \times 10^{3}} \right)}} \\{= {3.47 \times 10^{- 4}\mspace{11mu}(M)}}\end{matrix} & \left( {{Formula}\mspace{20mu} 3} \right)\end{matrix}$

This concentration is equivalent to the concentration of SPDP introducedinto CGG. Also, since the 2-pyridyldisulfide group of SPDP contributesto the absorbance at 280 nm, calculation of the CGG concentrationrequires the following correction. The absorbance at 280 nm attributedto CGG (A280, CGG) can be calculated by formula (4) below, where themolecular absorbance coefficient of a 2-pyridyldisulfide group at 280 nmis 5.1×10³.

$\begin{matrix}\begin{matrix}{\left( {{A280},{CGG}} \right) = {3.73 - \left( {3.4 \times 10^{- 4} \times 5.1 \times 10^{3}} \right)}} \\{= 1.96}\end{matrix} & \left( {{Formula}\mspace{20mu} 4} \right)\end{matrix}$

Therefore, the CGG concentration [CGG], and the number of SPDP moleculesintroduced into one CGG molecule [SPDP]/[CGG] can be calculated byformula (5) below, where the molar absorbance coefficient of CGG at 280nm is 1.99×10⁵.

$\begin{matrix}\begin{matrix}{\lbrack{CGG}\rbrack = {1.96/\left( {1.99 \times 10^{5}} \right)}} \\{= {9.85 \times 10^{- 6}\mspace{11mu}(M)}} \\{{\lbrack{SPDP}\rbrack/\lbrack{CGG}\rbrack} = {3.47 \times {10^{- 4}/\left( {9.85 \times 10^{- 6}} \right)}}} \\{= 35.2}\end{matrix} & \left( {{Formula}\mspace{20mu} 5} \right)\end{matrix}$

2. Preparation of Conjugate of Peptide Fructose and CGG

40 mg (7.18×10⁻² mmol) of a pyridyldithio-derivative was added to 50 mlof CGG-SPDP/PBS solution, and stirred at 4° C. overnight. The resultantprecipitate was removed by centrifugation (10 minutes, 20,000 rpm), andthereafter, the absorbance (A343) was measured at 343 nm, with a resultof 3.52. The resultant supernatant was subjected to gel filtration usinga Sephadex G25 column (manufactured by Pharmacia, diameter 2 cm×length80 cm). As a result, 48 ml of a conjugate of peptide fructose and CGGwas obtained.

The number of peptide fructoses binding to one CGG molecule wasdetermined as follows. Assuming that the molecular absorbancecoefficient of released pyridine-2-thione at 343 nm is 8.08×10³ based onthe above-described result that A343 was 3.52 immediately after thereaction, the concentration [pyridine-2-thione] can be calculated byformula (6) below.

$\begin{matrix}\begin{matrix}{\left\lbrack {{pyridine}\text{-}2\text{-}{thione}} \right\rbrack = {3.52/\left( {8.08 \times 10^{3}} \right)}} \\{= {4.35 \times 10^{- 4}\mspace{14mu}(M)}}\end{matrix} & \left( {{Formula}\mspace{20mu} 6} \right)\end{matrix}$

This concentration is equivalent to the concentration of peptidefructose introduced into CGG. Since the CGG concentration was 1.40×10⁻⁵M, the number of peptide fructose molecule introduced into one CGGmolecule [peptide fructose]/[CGG] can be calculated by formula (7).

$\begin{matrix}\begin{matrix}{{\left\lbrack {{peptide}\mspace{14mu}{fructose}} \right\rbrack/\lbrack{CGG}\rbrack} = {4.35 \times {10^{- 4}/\left( {1.40 \times 10^{- 5}} \right)}}} \\{= 31.1}\end{matrix} & \left( {{Formula}\mspace{20mu} 7} \right)\end{matrix}$

Example 2 Preparation of Peptide Fructose

In this example, peptide fructose represented by formula (VI) below wasprepared by the following procedure.

1. Introduction of Cys Group Into Wang Resin (Solid Phase)

Fmoc-Cys(StBu)-Resin was prepared in a manner as described in Example 1.

2. Introduction of Thr Group Into Fmoc-Cys(StBu)-Resin

19.2 g (7.668 mmol on a Cys basis) Fmoc-Cys(StBu)-Resin was dispersed in50 ml of 20% piperidine/DMF, and stirred at room temperature for 10minutes. The solvent was removed by filtration and thereafter the 20%piperidine/DMF reaction was repeated. The resin was washed with 50 ml ofDMF three times, and a small amount of the resin was subjected to aninhydrin test in a methanol solution. As a result, it was confirmedthat the amino group was in a free state. The resin was dispersed in 20ml of DMF. To this solution, 9.719 g (19.17 mmol) offluorenylmethoxycarbonylthreonine pentafluorophenylester (hereinafterabbreviated as Fmoc-Thr-Opfp) was added, and stirred at room temperaturefor two hours. The resin was washed with 50 ml of DMF three times.Thereafter, a small amount of resin was subjected to a ninhydrin test ina methanol solution. As a result, it was confirmed that no free aminogroup existed. Thereafter, the resin was washed with chloroform threetimes, followed by drying under vacuum overnight to obtain 17.2 g of thedried resin (hereinafter abbreviated as Fmoc-Thr-Cys(StBu)-Resin).

The ratio of introduction of a Thr group into the resin was measured ina manner as described in Example 1. The ratio of introduction of a Thrgroup was 42%.

3. Introduction of Leu Group Into Fmoc-Thr-Cys(StBu)-Resin

15.2 g (6.444 mmol on a Cys basis) Fmoc-Thr-Cys(StBu)-Resin wasdispersed in 50 ml of 20% piperidine/DMF, and stirred at roomtemperature for 10 minutes. The solvent was removed by filtration andthereafter 20% piperidine/DMF reaction was repeated. The resin waswashed with 50 ml of DMF three times, and a small amount of the resinwas subjected to a ninhydrin test in a methanol solution. As a result,it was confirmed that the amino group was in a free state. The resin wasdispersed in 20 ml of DMF. To this solution, 8.361 g (16.11 mmol) offluorenylmethoxycarbonylleucine pentafluorophenylester (hereinafterabbreviated as Fmoc-Leu-Opfp) was added, and stirred at room temperaturefor two hours. The resin was washed with 50 ml of DMF three times.Thereafter, a small amount of resin was subjected to a ninhydrin test ina methanol solution. As a result, it was confirmed that no free aminogroup existed. Thereafter, the resin was washed with chloroform threetimes, followed by drying under vacuum overnight to obtain 13.4 g of thedried resin (hereinafter abbreviated as Fmoc-Leu-Thr-Cys(StBu)-Resin).

The ratio of introduction of a Leu group into the resin was measured ina manner as described above. The ratio of introduction of a Leu groupwas 26%.

4. Introduction of His Group Into Fmoc-Leu-Thr-Cys(StBu)-Resin

Fmoc-His(Trt)-Leu-Thr-Cys(StBu)-Resin was prepared in a manner asdescribed in Example 1.

5. Introduction of Val Group Into Fmoc-His(Trt)-Leu-Thr-Cys(StBu)-Resin

Fmoc-Val-His(Trt)-Leu-Thr-Cys(StBu)-Resin was prepared in a manner asdescribed in Example 1.

6. Removal of Peptide Chain From Resin

Val-His-Leu-Thr-Cys was prepared in a manner as described in Example 1.

7. Introduction of Fructose Into Peptide Chain Due to AmadoriRearrangement Reaction

Fructose-Val-His-Leu-Thr-Cys was prepared in a manner as described inExample 1.

(Preparation of Peptide Fructose-Protein Conjugate)

In this example, CGG, BSA and KLH were used as proteins.

1. Preparation of CGG-Cys-Thr-Leu-His-Val-Fructose

200 mg (1.33×10⁻³ mmol) of CGG was dissolved in about 10 ml of PBS. Tothis mixture, a solution, in which 20.9 mg (0.0667 mmol, 50 eq.) of SPDPwas dissolved in 1 ml of ethanol, was gradually added at roomtemperature while stirring. After stirring for 30 minutes, the mixturewas subjected to purification using a Sephadex G25M column to obtain 24ml (2.07 mg/ml) of CGG-SPDP solution. The number of SPDPs introducedinto CGG was measured in the following manner.

0.5 ml of CGG-SPDP solution was collected, and the absorbance at 280 nm(A280) was measured, with a result of 6.03. Thereafter, 25 μl of 100 mMdithiothreitol solution was added to the CGG-SPDP solution, and themixture was allowed to stand for 3 minutes. Thereafter, the absorbanceat 343 nm (A343) was measured, with a result of 4.67. The 343-nmabsorbance was attributed to pyridine-2-thione released due to DTTreduction. The amount of pyridine-2-thione is equivalent to the amountof the introduced SPDP. Therefore, the SPDP concentration [SPDP] can becalculated by formula (8) below, where the molar absorbance coefficientof pyridine-2-thione at 343 nm is 8080.

$\begin{matrix}\begin{matrix}{\lbrack{SPDP}\rbrack = {4.67/8080}} \\{= {5.780 \times 10^{- 4}\mspace{14mu}(M)}}\end{matrix} & \left( {{Formula}\mspace{20mu} 8} \right)\end{matrix}$

280-nm absorption is attributed to the protein. The introduced SPDPabsorbs 280-nm light. Therefore, calculation of the CGG concentration[CGG] requires correction represented by formula (9) below, where themolar absorbance coefficient of SPDP at 280 nm is 5100, the molarabsorbance coefficient of CGG at 280 nm is 1.99×10⁵, (A280, SPDP)represents absorbance at 280 nm attributed to SPDP, and (A280, CGG)represents absorbance at 280 nm attributed to CGG.

$\begin{matrix}\begin{matrix}{\left( {{A280},{SPDP}} \right) = {5.780 \times 10^{- 4} \times 5100}} \\{= 2.95} \\{\left( {{A280},{CGG}} \right) = {6.03 - 2.95}} \\{= 3.08} \\{\lbrack{CGG}\rbrack = {3.08/\left( {1.99 \times 10^{5}} \right)}} \\{= {1.55 \times 10^{- 5}\mspace{14mu}(M)}}\end{matrix} & \left( {{Formula}\mspace{20mu} 9} \right)\end{matrix}$

Therefore, the number of SPDP molecules introduced into one CGG molecule[SPDP]/[CGG] is indicated by formula (10) below.[SPDP]/[CGG]=37.3  (Formula 10)

Into a PBS solution of the resultant CGG-SPDP (2.32 mg/ml, 13.5 ml),11.7 mg (0.016 mmol, 2 eq. SPDP) of fructose-Val-His-Leu-Thr-Cysdissolved in 0.1 ml of PBS was dropped at room temperature whilestirring. The mixture was mildly stirred at room temperature for 3hours. The reaction was tracked by measuring the absorbance of thereaction solution at 343 nm. The reaction solution was subjected topurification using a Sephadex G25M column to obtain 32 ml (0.885 mg/ml)of CGG-Cys-Thr-Leu-His-Val-fructose. Hereinafter, this peptidefructose-protein conjugate is abbreviated as F-CGG.

2. Preparation of KLH-Cys-Thr-Leu-His-Val-Fructose

57.8 mg (5.78×10⁻⁴ mmol) of KLH was dissolved in about 10 ml of PBS. Tothis mixture, a solution, in which 2.7 mg (0.00867 mmol, 15 eq.) of SPDPwas dissolved in 1 ml of ethanol, was gradually added at roomtemperature while stirring. After stirring for 30 minutes, the mixturewas subjected to purification using a Sephadex G25M column to obtain 28ml (1.47 mg/ml) of KLH-SPDP solution. The number of SPDPs introducedinto KLH was measured in the following manner.

0.5 ml of KLH-SPDP solution was collected, and the absorbance at 280 nm(A280) was measured, with a result of 1.88. Thereafter, 25 μl of 100 mMdithiothreitol solution was added to the CGG-SPDP solution, and themixture was allowed to stand for 3 minutes. Thereafter, the absorbanceat 343 nm (A343) was measured, with a result of 0.365. The 343-nmabsorbance was attributed to pyridine-2-thione released due to DTTreduction. The amount of pyridine-2-thione is equivalent to the amountof the introduced SPDP. Therefore, the SPDP concentration [SPDP] can becalculated by formula (11) below, where the molar absorbance coefficientof pyridine-2-thione at 343 nm is 8080.

$\begin{matrix}\begin{matrix}{\left\{ {SPDP} \right\rbrack = {0.365/8080}} \\{= {4.517 \times 10^{- 5}\mspace{14mu}(M)}}\end{matrix} & \left( {{Formula}\mspace{14mu} 11} \right)\end{matrix}$

280-nm absorption is attributed to the protein. The introduced SPDPabsorbs 280-nm light. Therefore, calculation of the KLH concentration[KLH] requires correction represented by formula (12) below, where themolar absorbance coefficient of SPDP at 280 nm is 5100, the molarabsorbance coefficient of KLH at 280 nm is 1.12×10⁵, and (A280, KLH)represents absorbance at 280 nm attributed to KLH.

$\begin{matrix}\begin{matrix}{\left( {{A\; 280},{SPDP}} \right) = {4.517 \times 10^{- 5} \times 5100}} \\{= 0.230} \\{\left( {{A\; 280},{KLH}} \right) = {1.88 - 0.230}} \\{= 1.65} \\{\lbrack{KLH}\rbrack = {1.65/\left( {1.12 \times 10^{5}} \right)}} \\{= {1.47 \times 10^{- 5}(M)}}\end{matrix} & \left( {{Formula}\mspace{14mu} 12} \right)\end{matrix}$

Therefore, the number of SPDP molecules introduced into one KLH molecule[SPDP]/[KLH] is indicated by formula (13) below.[SPDP]/[KLH]=3.07  (Formula 13)

Into a PBS solution of the resultant KLH-SPDP (2.65 mg/ml, 15.5 ml),8.81 mg (0.012 mmol, 10 eq. SPDP) of fructose-Val-His-Leu-Thr-Cysdissolved in 0.1 ml of PBS was dropped at room temperature whilestirring. The mixture was mildly stirred at room temperature for 3hours. The reaction was tracked by measuring the absorbance of thereaction solution at 343 nm. The reaction solution was subjected topurification using a Sephadex G25M column to obtain 32 ml (1.13 mg/ml)of KLH-Cys-Thr-Leu-His-Val-fructose. Hereinafter, this peptidefructose-protein conjugate is abbreviated as F-KLH.

3. Preparation of BSA-Cys-Thr-Leu-His-Val-Fructose

200 mg (0.00303 mmol) of BSA was dissolved in about 10 ml of PBS. To themixture, a solution, in which 4.73 mg (0.0152 mmol, 5 eq.) of SPDP wasdissolved in 1 ml of ethanol, was gradually added at room temperaturewhile stirring. After stirring for 30 minutes, the mixture was subjectedto purification using a Sephadex G25M column to obtain 16 ml (10.4mg/ml) of BSA-SPDP solution. The number of SPDPs introduced into BSA wasmeasured in the following manner.

0.5 ml of BSA-SPDP solution was collected, and the absorbance at 280 nm(A280) was measured, with a result of 9.18. Thereafter, 25 μl of 100 mMdithiothreitol solution was added to the BSA-SPDP solution, and themixture was allowed to stand for 3 minutes. Thereafter, the absorbanceat 343 nm (A343) was measured, with a result of 3.63. The 343-nmabsorbance was attributed to pyridine-2-thione released due to DTTreduction. The amount of pyridine-2-thione is equivalent to the amountof the introduced SPDP. Therefore, the SPDP concentration [SPDP] can becalculated by formula (14) below, where the molar absorbance coefficientof pyridine-2-thione at 343 nm is 8080.

$\begin{matrix}\begin{matrix}{\lbrack{SPDP}\rbrack = {3.63/8080}} \\{= {4.493 \times 10^{- 4}(M)}}\end{matrix} & \left( {{Formula}\mspace{14mu} 14} \right)\end{matrix}$

280-nm absorption is attributed to the protein. The introduced SPDPabsorbs 280-nm light. Therefore, calculation of the BSA concentration[BSA] requires correction represented by formula (15) below, where themolar absorbance coefficient of SPDP at 280 nm is 5100, the molarabsorbance coefficient of BSA at 280 nm is 4.36×10⁴, and (A280, BSA)represents absorbance at 280 nm attributed to BSA.

$\begin{matrix}\begin{matrix}{\left( {{A\; 280},{SPDP}} \right) = {4.493 \times 10^{- 4} \times 5100}} \\{= 2.29} \\{\left( {{A\; 280},{BSA}} \right) = {9.18 - 2.29}} \\{= 6.89} \\{\lbrack{BSA}\rbrack = {6.89/\left( {4.36 \times 10^{4}} \right)}} \\{= {1.58 \times 10^{- 4}(M)}}\end{matrix} & \left( {{Formula}\mspace{14mu} 15} \right)\end{matrix}$

Therefore, the number of SPDP molecules introduced into one BSA molecule[SPDP]/[BSA] is indicated by formula (16) below.[SPDP]/[BSA]=2.8  (Formula 16)

Into a PBS solution of the resultant BSA-SPDP (10.4 mg/ml, 16 ml), 10.3mg (0.014 mmol, 2 eq. SPDP) of fructose-Val-His-Leu-Thr-Cys dissolved in0.1 ml of PBS was dropped at room temperature while stirring. Themixture was mildly stirred at room temperature for 3 hours. The reactionwas tracked by measuring the absorbance of the reaction solution at 343nm. The reaction solution was subjected to purification using a SephadexG25M column to obtain 24 ml (6.51 mg/ml) ofBSA-Cys-Thr-Leu-His-Val-fructose. Hereinafter, this peptidefructose-protein conjugate is abbreviated as F-BSA.

(Preparation of Antiserum and Evaluation of its Performance)

Among the prepared peptide fructose-protein conjugates, F-CGG and F-KLHwere used as immunogens to prepare an antiserum by the method below.Combinations of immunogens and animals to be immunized used in preparingantisera are shown in Table 2.

TABLE 2 Immunization Method Age (weeks) Number of (at first ImmunogenAnimal Sex animals immunization) F-CGG Mouse ♀ 5 7 (BALB/C) F-KLH Mouse♀ 5 7 (A/J)

In immunization, F-CGG or F-KLH was mixed with complete Freund'sadjuvant (CFA) or incomplete Freund's adjuvant (IFA) using ahomogenizer. The mixture was injected into animals to be immunized. Thefinal protein concentration of the mixture was 1 mg/ml. The twoimmunogens were used to perform immunization in accordance with theimmunizing schedule shown in FIG. 1. Antisera collected 7 weeks afterfirst immunization were assessed for their performance using ELISA.Conditions for ELISA are shown in Table 3. The results of assessmentwhen the immunogen was F-CGG are shown in FIG. 2. The results ofassessment when the immunogen was F-KLH are shown in FIG. 3. In FIGS. 2and 3, the horizontal axis represents the dilution of the serum, and thevertical axis represents absorbance at a measurement wavelength of 492nm. Note that in FIGS. 2 and 3, a measurement point at the infinity ofthe serum dilution shows the measurement result of a sample which didnot contain serum.

TABLE 3 Conditions for ELISA Antigen coating: F-BSA, 0.1 mg/ml, 100μl/well, r.t., overnight Blocking: 1% BSA.PBS.Az, 200 μl/well, r.t., 30minutes Primary antibody: a dilution series of each antiserum (1%BSA.PBS.Az), r.t., 3 hours Secondary antibody: anti-mouse IgG-POD oranti-mouse IgM-POD, 0.2 μg/ml 1% BSA.PBS, r.t., 30 minutes Enzymaticreaction: o-phenylenediamine, 4 mg/ml PCB + 30% H₂O₂, r.t., 3 minutesTermination of reaction: 4NH₂SO₄, 25 μl/well

The titers of the antiserum obtained from F-CGG as an immunogen and theantiserum obtained from F-KLH as an immunogen were both observed at aserum dilution of 10⁵ to 10⁶. Since the amount of produced IgG waslarger than that of produced IgM, F-CGG and F-KLH have good performance.

Example 3 Preparation of Monoclonal Antibody

In this example, CGG and KLH were used as proteins for preparingconjugates employed as immunogens. As a myeloma-derived cell,p3×63·Ag8·653 was used. ELISA was adopted as an assessment method forcell screening. A conjugate containing BSA as a protein was prepared andused as a control for ELISA.

(Preparation of Conjugate to Be Used as Immunogen)

1. Preparation of pyridyldithiopropionylated CGG (Preparation ofCGG-SPDP)

200 mg (1.33×10⁻³ mmol) of CGG was dissolved in 50 ml of phosphatebuffer saline (hereinafter abbreviated as PBS). 5 ml of SPDP/ethanolsolution (SPDP=40 mg, 0.13 mmol, an equivalent weight of 100) wasdropped into the mixture while stirring. After stirring at roomtemperature for 30 minutes, the resultant precipitate was removed bycentrifugation (10 minutes, 20,000 rpm). The resultant supernatant wassubjected to gel filtration using a Sephadex G25M column (manufacturedby Pharmacia, diameter 2 cm×length 80 cm) to obtain 80 ml ofCGG-SPDP/PBS solution.

The number of SPDPs binding to one CGG molecule was determined asfollows. 1 ml of the resultant CGG-SPDP PBS solution was used and theabsorbance thereof was measured at 280 nm. The absorbance was 3.73.

50 μl of 100 mM dithiothreitol (hereinafter abbreviated as DTT) aqueoussolution was added to the CGG-SPDP/PBS solution, and was allowed tostand for 5 minutes. Thereafter, the absorbance was measured at 343 nm,with a result of 2.80.

Assuming that the molecular absorbance coefficient of pyridine-2-thionereleased by DTT reduction at 343 nm was 8.08×10³, the concentration[pyridine-2-thione] was determined by formula (A).

$\begin{matrix}\begin{matrix}{\left\lbrack {{pyridine}\text{-}2\text{-}{thione}} \right\rbrack = {2.80/\left( {8.08 \times 10^{3}} \right)}} \\{= {3.47 \times 10^{- 4}(M)}}\end{matrix} & \left( {{Formula}\mspace{20mu} A} \right)\end{matrix}$

This concentration is equivalent to the concentration of SPDP introducedinto CGG. Also, since the 2-pyridyldisulfide group of SPDP contributesto the absorbance at 280 nm, calculation of the CGG concentrationrequires the following correction. The absorbance at 280 nm attributedto CGG (A_(280, CGG)) can be calculated by formula (B) below, where themolecular absorbance coefficient of a 2-pyridyldisulfide group at 280 nmis 5.1×10³.

$\begin{matrix}\begin{matrix}{A_{280,{CGG}} = {3.73 - \left( {3.47 \times 10^{- 4} \times 5.1 \times 10^{3}} \right)}} \\{= 1.96}\end{matrix} & \left( {{Formula}\mspace{20mu} B} \right)\end{matrix}$

Therefore, the CGG concentration [CGG], and the number of SPDP moleculesintroduced into one CGG molecule [SPDP]/[CGG] can be calculated byformula (C) below, where the molar absorbance coefficient of CGG at 280nm is 1.99×10⁵.

$\begin{matrix}\begin{matrix}{\lbrack{CGG}\rbrack = {1.96/\left( {1.99 \times 10^{5}} \right)}} \\{= {9.85 \times 10^{- 6}(M)}} \\{{\lbrack{SPDP}\rbrack/\lbrack{CGG}\rbrack} = {3.47 \times {10^{- 4}/\left( {9.85 \times 10^{- 6}} \right)}}} \\{= 35.2}\end{matrix} & \left( {{Formula}\mspace{20mu} C} \right)\end{matrix}$

2. Preparation of Conjugate of Peptide Fructose and CGG

40 mg (7.18×10⁻² mmol) of a pyridyldithio-derivative was added to 50 mlof CGG-SPDP/PBS solution, and stirred at 4° C. overnight. The resultantprecipitate was removed by centrifugation (10 minutes, 20,000 rpm), andthereafter, the absorbance (A₃₄₃) was measured at 343 nm, with aresulting of A₃₄₃=3.52. The resultant supernatant was subjected to gelfiltration using a Sephadex G25 column (manufactured by Pharmacia,diameter 2 cm×length 80 cm). As a result, 48 ml of a conjugate ofpeptide fructose and CGG was obtained.

The number of peptide fructoses binding to one CGG molecule wasdetermined as follows. Assuming that the molecular absorbancecoefficient of released pyridine-2-thione at 343 nm is 8.08×10³ based onthe above-described result that A₃₄₃ was 3.52 immediately after thereaction, the concentration [pyridine-2-thione] can be calculated byformula (D) below.

$\begin{matrix}\begin{matrix}{\left\lbrack {{pyridine}\text{-}2\text{-}{thione}} \right\rbrack = {3.52/\left( {8.08 \times 10^{3}} \right)}} \\{= {4.35 \times 10^{- 4}(M)}}\end{matrix} & \left( {{Formula}\mspace{20mu} D} \right)\end{matrix}$

This concentration is equivalent to the concentration of peptidefructose introduced into CGG. Since the CGG concentration was 1.40×10⁻⁵M, the number of peptide fructose molecules introduced into one CGGmolecule [peptide fructose]/[CGG] can be calculated by formula (E).

$\begin{matrix}\begin{matrix}{{\left\lbrack {{peptide}\mspace{14mu}{fructose}} \right\rbrack/\lbrack{CGG}\rbrack} = {4.35 \times {10^{- 4}/1.40} \times 10^{- 5}}} \\{= 31.1}\end{matrix} & \left( {{Formula}\mspace{20mu} E} \right)\end{matrix}$

3. Preparation of Conjugate of KLH and Peptide Fructose

54 mg of KLH and 5.42 mg (30 eq.) of SPDP were used to preparepyridyldithiopropionylated KLH (KLH-SPDP) in a manner as described aboveconcerning CGG, with a result of 28 ml of KLH-SPDP/PBS solution(concentration: 1.47 mg/ml). The number of SPDPs introduced into one KLHmolecule was 3.0.

15.5 ml (41.1 mg) of the resultant KLH-SPDP solution was used to reactwith peptide fructose (6.4 mg) as described above, with a result of 33.3mg (3.33 mg/ml, 10 ml) of a conjugate of KLH and peptide fructose. Thenumber of peptide fructoses introduced into one KLH molecule was 2.7.

4. Preparation of Conjugate of BSA and Peptide Fructose

A conjugate of peptide fructose and BSA was prepared as a control forELISA. 200 mg of BSA and 4.73 mg (5 eq.) of SPDP were used to preparepyridyldithiopropionylated BSA (BSA-SPDP) in a manner as described aboveconcerning CGG and KLH, with a result of 16 ml of BSA-SPDP/PBS solution(concentration: 10.4 mg/ml). The number of SPDPs introduced into one BSAmolecule was 2.8.

16 ml of the resultant BSA-SPDP solution was used to react with peptidefructose (7.4 mg) in a manner as described above, with a result of 156.2mg (6.51 mg/ml, 24 ml) of a conjugate of peptide fructose and BSA. Thenumber of peptide fructoses introduced into one BSA molecule was 2.5.

(Method of Preparing Monoclonal Antibody-Producing Cells and MonoclonalAntibody)

1. Immunization of Mice

Ten about 8-week old mice (Balb/c) were divided into two groups (fivefor each). Two immunogens (CGG conjugate and KLH conjugate) as preparedabove were intraperitoneally injected into the respective groups at adose of 100 μL.

2. Check of Antibody Production

50 to 100 μL of blood was collected into a centrifuge tube from theophthalmic vein of mice, which were bled 77 days after injection forimmunization. The sera were centrifuged, followed by assessment ofantibody titer using ELISA (described below). As a result, production ofanti-HbA1c antibodies was confirmed in all of the mice.

3. Booster for Mice

Booster injection of a weak immunogen was performed for mice having aparticularly high titer in the above-described antibody titer assessmentin order to enlarge the spleens of the mice (the group of mice given theCGG conjugates as an immunogen). The immunogen was 1 mg/mL solution, inwhich the CGG conjugate with fructose was diluted in PBS, without anyadjuvant.

4. Cell Fusion

Three days after the booster, spleen cells were isolated from the mice.The spleen cells were fused with a mouse myeloma-derived cell line(P3×63−Ag8.653) by a commonly used method using polyethylene glycolhaving an average molecular weight of 1,500. Spleen cells from the samemice were used as feeder cells (cells supplying a growth factor). Thefused cells were cultured in HAT medium containing 15% fetal calf serum(hereinafter abbreviated as FCS) on two 96-well plates. After one week,the medium was exchanged with HT medium containing 15% FCS.

5. Cloning

Antibody titers were measured using ELISA. The 5 wells having thehighest titers were selected.

The media in the 5 wells were diluted and dispensed on five 96-wellplates at a density of one cell per well (limiting dilution). Thymuscells from 5 week old mice (Balb/c) were used as feeder cells to promoteinitial growth. The cells were further cultured while increasing thesize of the plate. The supernatant was subjected to ELISA so as tomeasure antibody titers at appropriate times. A group of cells, whichexhibited a high titer to HbA1c and good growth, were finally selected,and were cultured to a concentration of 5×10⁵ cells/mL in 200 ml.

6. The supernatant of the finally selected cells was centrifuged. Thecells were suspended in 1 ml of solution containing FCS anddimethylsulfoxide (9:1) at a concentration of 5×10⁶ cells/mL. Thesuspension was frozen at −80° C. and was transferred to liquid nitrogenfor long-term preservation.

7. A monoclonal antibody was purified from the supernatant of theculture medium by affinity chromatography using2-mercaptopyridine-binding gel (HiTrap IgM Purification, manufactured byPharmacia). The monoclonal antibody was confirmed to be IgM according tothe test using a Mouse Monoclonal Typing Kit (manufactured by BindingSite).

(Measurement of Antibody Titer by ELISA)

The antisera and culture supernatants obtained in the preparation of theabove-described antibody were assessed by ELISA. Conditions and methodsfor the measurement are described below.

1. Antigen Coating

A conjugate of peptide fructose and BSA was diluted with PBS containing0.1 mg/ml and 0.04% sodium azide (BSA•PBS•Az) to prepare an antigensolution having a BSA concentration of 0.1 mg/mL. 100 μL of the antigensolution was plated per well in microplates (vinyl chloride 96-wellplates manufactured by Costar), and allowed to stand at 20° C.overnight. An aspirator was used to remove the antigen solution, andthereafter, the microplates were washed with PBS three times. Theremaining PBS was removed by an aspirator.

Purified HbA1c and purified HbA0 were optionally used as antigens forcoating depending on the measurement. In this case, the antigenconcentration was 0.02 mg/ml, and after plating into wells, the antigensolution was allowed to stand at 4° C. overnight.

2. Blocking

200 μL of 1% BSA•PBS•Az was plated per well, and allowed to stand for 30minutes at room temperature. Thereafter, BSA•PBS•Az was removed using anaspirator, and the plates were washed with PBS three times. Ifsubsequent procedures were not performed on the same day, the platesremaining in this state were preserved at 4° C. along with a filterwetted with water.

3. Reaction of Antibody

100 μL of antibody solution (a serum, a culture supernatant, a purifiedantibody, or the like), diluted to various dilutions with 1% BSA•PBS•Az,was plated per well of the plates prepared with the above-describedprocedures. The plates were allowed to stand at room temperature for 3hours. Thereafter, the antibody solution was removed using an aspirator.The plates were washed with PBS three times. The remaining PBS wasremoved using an aspirator.

4. Reaction of Secondary Antibody

0.2 μg/mL peroxidase-labeled goat anti-mouse IgG or IgM antibody(manufactured by KPL) was dissolved in 1% BSA/PBS solution (secondaryantibody solution). 100 μL of the secondary antibody solution was platedper well of plates and allowed to stand at room temperature for 30minutes. An aspirator was used to remove the secondary antibodysolution. The plates were washed with PBS three times. The remaining PBSwas removed using an aspirator.

5. Reaction of Substrate and its Termination

40 mg of o-phenylenediamine (for biochemistry) was dissolved in 10 mL ofcitrate-phosphate buffer (pH 5). 4 μL of 30% hydrogen peroxide was addedto the mixture immediately before use (substrate solution). 100 μL ofthe substrate solution was plated per well and allowed to stand at roomtemperature. After 3 to 5 minutes, 25 μl of 4N sulfuric acid was addedper well to terminate the reaction.

6. Measurement

Absorbance was measured at 492 nm using a microplate reader (manufactureby Tosoh Corporation).

(Results of Measurement of Antibody Titers)

The results of measurement of the antibody titer of an antiserumcollected immediately before cell fusion (77 days after immunization)according to the above-described methods are shown in FIG. 4. Theresults of measurement of the antibody titers of monoclonal antibodiesobtained by purifying the culture supernatants of two clones (4F and 8E)selected by cloning are shown in FIG. 5 (4F) and FIG. 6 (8E).

The result of FIG. 4 shows that 77 days after immunization, theantiserum had capability of binding similarly to BSA-peptide fructoseand HbA1c, and substantially no capability of binding to HbA0.Therefore, it is believed that this antiserum recognizes the fructosebinding site of HbA1c as originally aimed.

Further, it is indicated that the two clones selected by cloning aftercell fusion (4F and 8E) have a binding ability of 1×10⁻⁹ M and 3×10⁻⁹ M,respectively at the half value of the binding ability.

Deposit

Two hybridomas obtained in the present invention were deposited with theInternational Patent Organism Depository, the National Institute ofAdvanced Industrial Science and Technology (address: AIST TsukubaCentral 6, 1-1, Higashi, 1-Chome Tsukuba-shi, Ibaraki-ken, 305-8566Japan) on Jun. 22, 2001 under designation FERM BP-7637 and FERM BP-7636.

INDUSTRIAL APPLICABILITY

As described above, an —SH group is introduced into the peptide fructoseof the present invention in addition to fructose-valine-histidine whichis a characteristic structure of HbA1c. Therefore, it is easy for thepeptide fructose to be linked to a protein. By linking the peptidefructose to a protein, a protein conjugate can be produced.

The peptide fructose-protein conjugate of the present invention hasfructose-valine-histidine which is a characteristic structure of HbA1cand a protein other than hemoglobin. Therefore, this protein conjugatehas no structure common to hemoglobins other than HbA1c. Therefore, byusing this protein conjugate as an immunogen, anti-HbA1c antibodieswhich do not have cross reactivity to HbA0, and preferably do not havecross reactivity to hemoglobin other than HbA1c, can be reliablyproduced.

The present invention relates to a monoclonal antibody-producing cellobtained by fusing a mouse spleen cell, which has been sensitized with aconjugate of peptide fructose including a formula (I) and a protein,with a myeloma-derived cell line, followed by cloning, and a monoclonalantibody produced by the monoclonal antibody-producing cell. Therefore,it is possible to provide a monoclonal antibody specific to HbA1c inhuman hemoglobin, and an antibody-producing cell producing themonoclonal antibody.

1. A protein conjugate, comprising a peptide fructose linked with aprotein, and the peptide fructose compound is represented by formula (I)below:

wherein R1 represents a cysteine residue, the R1 is linked to thecarboxy-terminus of R2 at (b) with a covalent bond, the R2 contains twoor more amino acids derived from the amino acid sequence of HbA1 or anamino acid analog functionally equivalent to the amino acids, and the R2is linked to the fructose at the amino-terminus thereof, and the proteinis CGG, wherein the peptide fructose compound is linked to CGG at R1. 2.A protein conjugate according to claim 1, wherein the R2 contains atleast a peptide represented by formula (II) below:


3. A protein conjugate according to claim 1, wherein the R2 contains atleast a peptide represented by formula (III) below:


4. A protein conjugate according to claim 1, wherein the covalent bondat (b) is a peptide bond.
 5. An antiserum, produced in the blood of ananimal by injecting a protein conjugate according to claim 1 into saidanimal.
 6. An antibody, isolated from an antiserum according to claim 5.7. An isolated monoclonal antibody-producing cell, wherein themonoclonal antibody-producing cell is obtained by fusing a spleen cellof a mouse sensitized with a protein conjugate according to claim 1,with a myeloma-derived cell, and cloning a fused cell, and themonoclonal antibody-producing cell produces a monoclonal antibodycapable of specifically binding to human hemoglobin A1c.
 8. Themonoclonal antibody-producing cell according to claim 7, designated asdeposit number FERM BP-7637 or FERM BP-7636.
 9. A method of producing amonoclonal antibody-producing cell capable of producing a monoclonalantibody capable of specifically binding to human hemoglobin A1c, themethod comprising the steps of: a) sensitizing a mouse with a proteinconjugate according to claim 1; and b) isolating a spleen cell from thesensitized mouse, and fusing the spleen cell with a myeloma-derivedcell.
 10. A monoclonal antibody, produced by a monoclonalantibody-producing cell according to claim 7, which specifically bindsto human hemoglobin A1c.
 11. A monoclonal antibody produced by themonoclonal antibody-producing cell sensitized with a protein conjugateaccording to claim 1, wherein the binding constant to human hemoglobinA1c thereof is 10⁴ or more.